Formaldehyde is known to be a cytotoxin rich in reactivity with protein, membrane, and DNA, which causes various disorders by being inhaled or oral administration. Formaldehyde contained in atmosphere, waste water, and food has been considered problematic in recent years, and a convenient method for accurately measuring formaldehyde has been demanded.
As a determination method of formaldehyde, a method comprising calorimetric analysis using Hanz reagent, CTA reagent (J. Biol. Chem., 231, 813 (1958)), Purpald reagent (Anal. Biochem., 234(1), 50 (1996)) and an analysis method using a reagent containing phenylhydrazine, potassium ferricyanide, chloroform and methanol in combination are known. These methods are associated with problems in that they require complicated manipulation and a long time for the determination, or use of harmful reagents. As a means for solving such problems, enzymatic methods using glutathione-independent formaldehyde dehydrogenase (EC 2.1.1.46) have been disclosed (JP 5-42000 A, JP 2000-225000 A). These enzymatic methods analyze reduced nicotinamide adenine dinucleotide simultaneously produced when forming formic acid from formaldehyde, based on the enzymatic reaction or a pigment produced by the reaction of the reduced nicotinamide adenine dinucleotide. The determination sensitivity is, nevertheless, not necessarily sufficient for the measurement of a trace amount of formaldehyde, because it depends on the molar absorption coefficient of the reduced nicotinamide adenine dinucleotide or pigment.
As a highly sensitive assay of a trace amount of a substance by the use of an enzyme, a method is known wherein the determination object substrate and a coenzyme of an enzyme acting on a substrate are amplified by cycling reaction and quantitatively measured (Kensa to Gijutsu, 27(8), July, 1999). As one of the cycling methods, a determination method by an enzyme cycling method utilizing a reversible reaction using dehydrogenase and two kinds of coenzymes (thio-NAD compound and NADH compound, or NAD compound and thio-NADH compound) has been reported (JP 6-61278 B, JP 6-73477 B, JP 6-73478 B, JP 6-73479 B, JP 3023700, JP 3034969, JP 3034979, JP 3034984, JP 3034986, JP 3034987, JP 3034988, JP 8-103298 A).
JP 4-341198 A discloses a highly sensitive assay of alcohols or aldehydes using alcohol dehydrogenase and thio-NAD compound and NADH compound, or NAD compound and thio-NADH compound. The representative enzyme of the alcohol dehydrogenase used here, which catalyzes the following reaction, is the enzyme of EC1.1.1.1:alcohol+NAD(P)+aldehyde+NAD(P)HD. Schomburg, D. Stephan Eds., Enzyme Handbook 9 (Springer-Verlag) states that this enzyme oxidizes not only methanol as a substrate but also its product (formaldehyde). Since formaldehyde exists as a hydrate in an aqueous solution and as alcohol, it is a substrate oxidized with alcohol dehydrogenase allowing oxidation to acetic acid. Thus, a highly sensitive determination of formaldehyde using this enzyme is unattainable. In addition, since an aldehyde dehydrogenase, such as glutathione-independent formaldehyde dehydrogenase, irreversibly oxidizes formaldehyde, it cannot be applied to a highly sensitive determination by a cycling reaction.
In the field of analytical science, some compounds are known to be measured via formaldehyde as an intermediate. Of these, useful is the determination of creatinine, creatine and homocysteine in clinical tests.
Creatinine is a major diagnostic marker of kidney function in clinical tests, and determination of creatine is used for the analysis of disease state of muscular dystrophy, hyperthyroidism. As the determination method therefor, the Jaffe method is dominantly used, but this method is pointed out to be insufficient in the specificity. Recently, enzymatic methods using creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase and peroxidase having high specificity are increasingly used, but it has been pointed out that these methods may be influenced by reducing substances present in the body. There are reported some enzymatic methods wherein glutathione-independent formaldehyde dehydrogenase is used instead of peroxidase to analyze formaldehyde derived from sarcosine oxidase reaction by the above-mentioned method (Clin. Clim. Acta, 122, 181 (1982), Ann. Clin. Biochem., 29, 523 (1992)). However, these methods are not necessarily sufficient for the measurement of a trace amount, as mentioned above.
Homocysteine is an amino acid having an SH group produced by metabolism of methionine which is an essential amino acid in the body and is generally present at a low concentration in the body. It is known that homocystinuria, which is a genetic disease of these metabolizing enzymes causing increased blood homocysteine concentrations, relates to arteriosclerosis. In recent years, the connection of homocysteine concentration of the level somewhat higher than the normal value, to cerebral infarction, myocardial infarction and depths vein thrombosis (so-called economy class syndrome) has been clarified, and the present understanding is that blood homocysteine concentration is an independent risk factor of these diseases. The standard method for determination of homocysteine has been heretofore a method using HPLC (Clin. Chem., 39, 1590 (1993)), and various modifications have been also reported. The method using HPLC requires sophisticated analyzing devices and is inconvenient for processing a number of test samples. As a method without separation by HPLC, a method for determining homocysteine has been proposed to react homocysteine with S-adenosylhomocysteinase in the presence of adenosine and fluorescein-labeled S-adenosylhomocysteine, and measure S-adenosylhomocysteine present in the reaction system by fluorescence polarization immunoassay using an anti-S-adenosylhomocysteine antibody (JP 8-506478 A). Recently proposed enzymatic methods include a method comprising reacting homocysteine with homocysteine desulfrase and measuring ammonia, α-keto acid or hydrogen sulfide produced thereby (JP 2000-502262 A), a method comprising measuring hydrogen sulfide or thiol compound-substituted homocysteine produced in the presence of thiol compound, using L-methionine γ-lyase and O-acetylhomoserine-lyase capable of decomposing homocysteine (JP 2000-166597 A), a method comprising quantitatively measuring γ-substituted-α-aminobutylic acid or hydrogen sulfide derived from homocysteine by a γ-substituted-α-aminobutyrate synthase in the presence of a nucleophilic reagent substitutable with γ-mercapto group of homocysteine (JP 2000-228998 A) However, it is a general understanding that immunoassay requires a longer time and higher cost than do typical biochemical tests, and analysis methods using enzymatic reactions are not entirely satisfactory for the accurate measurement of homocysteine amount, since the blood homocysteine is in a trace amount and its concentration is about 10 μM or lower by normal value, leading to insufficient determination sensitivity, or substrate specificity of the enzyme used causes cross-reaction with substances other than homocysteine in the test sample.
As a homocysteine determination method utilizing an enzyme cycling method, a method for measuring pyruvic acid and ammonia produced by cystathionine β-synthase and cystathionine β-lyase (U.S. Pat. No. 6,174,696), a method for analyzing NAD compound or thio-NAD compound produced or consumed using homocysteine desulfrase and 2-keto-butylate dehydrogenase are known. In view of the homocysteine amount in the body, however, a method capable of assaying with higher sensitivity is desired.
It is therefore an object of the present invention to enable highly sensitive and convenient determination of formaldehyde or a compound that produces formaldehyde as a reaction intermediate. The present invention also aims at providing a method for determining homocysteine highly sensitively and conveniently without using HPLC or immunoassay.